title: Transforming a fructan:fructan 6G-fructosyltransferase from perennial ryegrass into a sucrose:sucrose 1-fructosyltransferase.
abstract: Fructosyltransferases (FTs) synthesize fructans, fructose polymers accumulating in economically important cool-season grasses and cereals. FTs might be crucial for plant survival under stress conditions in species in which fructans represent the major form of reserve carbohydrate, such as perennial ryegrass (Lolium perenne). Two FT types can be distinguished: those using sucrose (S-type enzymes: sucrose:sucrose 1-fructosyltransferase [1-SST], sucrose:fructan 6-fructosyltransferase) and those using fructans (F-type enzymes: fructan:fructan 1-fructosyltransferase [1-FFT], fructan:fructan 6G-fructosyltransferase [6G-FFT]) as preferential donor substrate. Here, we report, to our knowledge for the first time, the transformation of an F-type enzyme (6G-FFT/1-FFT) into an S-type enzyme (1-SST) using perennial ryegrass 6G-FFT/1-FFT (Lp6G-FFT/1-FFT) and 1-SST (Lp1-SST) as model enzymes. This transformation was accomplished by mutating three amino acids (N340D, W343R, and S415N) in the vicinity of the active site of Lp6G-FFT/1-FFT. In addition, effects of each amino acid mutation alone or in combination have been studied. Our results strongly suggest that the amino acid at position 343 (tryptophan or arginine) can greatly determine the donor substrate characteristics by influencing the position of the amino acid at position 340. Moreover, the presence of arginine-343 negatively affects the formation of neofructan-type linkages. The results are compared with recent findings on donor substrate selectivity within the group of plant cell wall invertases and fructan exohydrolases. Taken together, these insights contribute to our knowledge of structure/function relationships within plant family 32 glycosyl hydrolases and open the way to the production of tailor-made fructans on a larger scale.
authors.affiliation: UMR INRA UCBN 950 EVA, Ecophysiologie Végétale, Agronomie et Nutritions NCS, Université de Caen, 14032 Caen cedex, France.
authors.forename: Bertrand
authors.initials: B
authors.lastname: Lasseur
authors.forename: Lindsey
authors.initials: L
authors.lastname: Schroeven
authors.forename: Willem
authors.initials: W
authors.lastname: Lammens
authors.forename: Katrien
authors.initials: K
authors.lastname: Le Roy
authors.forename: German
authors.initials: G
authors.lastname: Spangenberg
authors.forename: Hélène
authors.initials: H
authors.lastname: Manduzio
authors.forename: Rudy
authors.initials: R
authors.lastname: Vergauwen
authors.forename: Jérémy
authors.initials: J
authors.lastname: Lothier
authors.forename: Marie-Pascale
authors.initials: MP
authors.lastname: Prud'homme
authors.forename: Wim
authors.initials: W
authors.lastname: Van den Ende
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008129:Lolium / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity; D001426:Bacterial Proteins
chemical_list: D006602:Hexosyltransferases; D010940:Plant Proteins; C105700:fructan - fructan 6G-fructosyltransferase; C021451:inulosucrase; D001426:Bacterial Proteins